JP3893107B2 - Method for producing fatty acid - Google Patents

Description

【００４５】
【発明の効果】
本発明の製造方法によれば、PUFAを高濃度に含有する脂肪酸が、極めて効率よく高い収率で得られる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a fatty acid containing a highly unsaturated fatty acid at a high concentration in a short time with a high decomposition rate and a high yield.
[0002]
[Prior art]
In recent years, bioactivity of polyunsaturated fatty acids (PUFA) such as arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid (DHA) has attracted attention. In particular, DHA has the effect of inhibiting platelet aggregation, lowering blood triglycerides, It is known to have an effect of lowering mid-cholesterol, an anticancer effect, an improvement of brain function, and the like. For this reason, the industrial production method of fats and oils rich in PUFA has been energetically researched and developed. As a representative method, many methods for concentrating PUFA to the glyceride side by cleaving fatty acids other than PUFA using the fatty acid selectivity of lipase have been reported.
[0003]
On the other hand, producing PUFA itself as a free acid or a fatty acid containing this in a high concentration, not as a PUFA-rich glyceride, is also important for use in drug development. In this case as well, PUFA-containing fats and oils are hydrolyzed by lipase, but there are limitations in terms of degradation rate and PUFA concentration by ordinary means. For this reason, a technique for achieving a high decomposition rate and a high PUFA concentration by using two kinds of lipases has been proposed. For example, by using a lipase having triglyceride position selectivity and a lipase not having triglyceride position selectivity for the hydrolysis of PUFA, the hydrolysis rate is increased, and a fatty acid having a high yield and a high neutralization value is obtained. (See Patent Document 1), DHA-containing fats and oils are hydrolyzed using lipases derived from the genus Candida to obtain fats and oils with high DHA content, and then hydrolyzed using lipases derived from the genus Penicillium. However, there is a method of fractionating fatty acids containing DHA at a high concentration (see Patent Document 2).
[0004]
However, the method of Patent Document 1 has a problem that the PUFA content in the raw oil and fat is low, and the PUFA concentration in the fatty acid remains low even when the decomposition rate is increased. In the method of Patent Document 2, since the lipase used in the second hydrolysis is a so-called partial glyceride lipase that acts on monoglyceride or diglyceride but does not act on triglyceride, it is concentrated on triglyceride. There is a problem that DHA cannot be used and a high yield cannot be obtained.
[0005]
[Patent Document 1]
Japanese Patent Laid-Open No. 60-130396 [Patent Document 2]
Japanese Patent Laid-Open No. 07-51075 [0006]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a method for producing a fatty acid containing a highly unsaturated fatty acid at a high concentration in a short time with a high decomposition rate and a high yield.
[0007]
[Means for Solving the Problems]
The present inventor hydrolyzes PUFA-containing fats and oils using lipase, separates free fatty acids to obtain high-PUFA-containing fats and oils, and further hydrolyzes them by simultaneously acting two specific lipases. As a result, it has been found that fatty acids containing PUFA in high concentration can be obtained very efficiently and in high yield.
[0008]
That is, the present invention provides a method for producing a PUFA-rich fatty acid comprising the following first step and second step.
[0009]
First step: A step of allowing a lipase that acts on triglycerides to act on a PUFA-containing fat and oil to hydrolyze, then separating free fatty acids to obtain a fat having an increased PUFA ratio.
Second step: A step of obtaining a fatty acid containing PUFA at a high concentration by hydrolyzing a lipase acting on triglyceride and a partial glyceride lipase simultaneously on the high fat content of PUFA obtained in the first step. [0011]
The polyunsaturated fatty acid (PUFA) referred to in the present invention means a fatty acid having 20 or more carbon atoms having 3 or more double bonds. Specifically, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA) and the like are preferable.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
[First step]
As the lipase used in the first step of the method of the present invention, a commercially available product can be used, but by fixing, the concentration rate of the PUFA-containing fat can be further increased, and the use of the lipase. The amount can also be reduced. Immobilized lipase is produced as follows.
[0013]
The carrier for immobilization used for immobilizing lipase is an inorganic carrier such as celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics; cellulose powder, polyvinyl alcohol, polypropylene, Examples thereof include organic polymers such as chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, and synthetic adsorption resin. Anion exchange resins, particularly porous anion exchange resins are preferred. Since such a porous carrier has a large surface area, a larger amount of enzyme adsorbed can be obtained. The particle diameter of the resin is preferably 100 to 1000 μm, and the pore diameter is preferably 100 to 1500 mm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene, and the like, and phenol formaldehyde resin (for example, Duolite A-568 manufactured by Rohm and Hass) is particularly preferable.
[0014]
Examples of lipases that act on triglycerides include the genus Candida, the genus Mucor, the genus Rhizopus, the genus Aspergillus, the genus Pseudomonas, and the genus Alcaligenes. Those derived from the genus Candida are particularly preferred. The amount of lipase used is 5 to 100%, particularly 10 to 50%, based on the weight of the carrier. At the time of immobilization, the lipase is in a solution state, but may be in a range that does not cause denaturation of the enzyme, and is preferably adjusted to pH 3 to 9 with a buffer solution. Moreover, the temperature at the time of immobilization is 0-60 degreeC, Especially 5-45 degreeC is preferable.
[0015]
After immobilizing the lipase, it is contacted with a fatty acid triglyceride or a fatty acid partial glyceride. Fatty acid triglycerides and fatty acid partial glycerides to be contacted with the immobilized lipase include vegetable liquid oils such as rapeseed oil, soybean oil, sunflower oil, fish oil such as sardine oil, tuna oil, bonito oil, sea animal oil such as whale oil, etc. Derived monoglycerides and diglycerides, mixtures thereof, and transesterified oils and fats obtained from these fats and oils can also be used. Two or more of these may be used in combination. The amount of fatty acid glyceride used is 100 to 3000%, particularly 250 to 2000% with respect to the weight of the carrier from the viewpoint of sufficient contact with the immobilized lipase and avoiding waste due to use of an excessive amount. preferable. The contact method may be any method such as immersion, stirring, or passing through a column filled with the immobilized enzyme with a pump or the like. The contact temperature is preferably 5 to 40 ° C., and the contact time is preferably 2 to 48 hours. When the contact is over, the mixture is filtered to recover the immobilized lipase. By such treatment, hydrolysis of fatty acid glycerides occurs, so that residual moisture in the immobilized lipase is consumed, and the amount of enzyme moisture can be reduced. Thus, it is considered that excess moisture is removed while maintaining moisture capable of maintaining the structure of the lipase, and the vicinity of the lipase is in a state suitable for the reaction. The above treatment is preferably carried out so that the water content of the immobilized lipase after filtration and recovery is in the range of 20 to 100%, particularly 40 to 70%, relative to the weight of the carrier.
[0016]
This immobilized lipase, unlike the immobilized lipase obtained by the usual preparation method through vacuum or reduced pressure drying, does not cause inactivation, but the high ultimate decomposition rate of fats and oils is the same as the high enzyme activity. Since it is irrelevant, it is surprising that a high decomposition rate as in the present invention can be obtained. As a result, even if oil and fat with a low PUFA content such as fish oil itself is used as a raw material, it can be directly subjected to hydrolysis without pretreatment to increase the PUFA content to some extent by wintering etc. Thus, the content of PUFA accumulated in glycerides can be increased to a level that has not been possible in the past.
[0017]
In order to increase the activity of the immobilized lipase, a treatment for adsorbing a fat-soluble fatty acid or a derivative thereof to a carrier in advance may be performed before the lipase is immobilized. Examples of the fat-soluble fatty acid to be used include saturated or unsaturated, linear or branched fatty acids having 8 to 18 carbon atoms, which may be substituted with a hydroxyl group. Specifically, capric acid, lauric acid, myristylic acid, oleic acid, linoleic acid, α-linolenic acid, ricinoleic acid, isostearic acid and the like can be mentioned. Examples of the derivatives include esters of these fatty acids with mono- or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoglycerides, diglycerides, ethylene oxide adducts thereof, polyglycerin esters, sorbitan esters, and sucrose esters of the above fatty acids. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.
[0018]
As a method for contacting these fat-soluble fatty acids or derivatives thereof and the carrier, these may be added directly to water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acids or derivatives thereof are once dispersed in the organic solvent. After dissolution, it may be added to a carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The amount of the fat-soluble fatty acid or derivative thereof used is preferably 1 to 500%, particularly preferably 10 to 200%, based on the weight of the carrier. The contact temperature is preferably 0 to 100 ° C., particularly preferably 20 to 60 ° C., and the contact time is preferably about 5 minutes to 5 hours. The carrier after this treatment may be dried. The drying temperature is preferably room temperature to 100 ° C, and drying under reduced pressure may be performed.
[0019]
In the first step, the lipase or the immobilized lipase is allowed to act on the PUFA-containing fat and oil for hydrolysis, and then free fatty acids are separated to produce a high-PUFA containing fat. As a specific embodiment of the reaction, a method of adding a lipase or an immobilized lipase to a PUFA-containing oil and fat, further adding water, and performing a hydrolysis reaction while stirring at a constant temperature, an immobilized lipase is added to a column (fixed bed). ), And a mixture of PUFA-containing fats and water is circulated through it.
[0020]
The PUFA-containing fat used for the hydrolysis may be any of triglycerides, diglycerides, monoglycerides and mixtures thereof containing PUFA such as DHA as part of the constituent fatty acids. In general, fish oil such as sardine oil, tuna oil and bonito oil, and sea animal oil such as whale oil can be mentioned. Commercially available DHA-containing fats and oils may also be used.
[0021]
The amount of lipase used in the reaction can be appropriately determined in consideration of the activity of lipase, but is preferably 0.3 to 30%, particularly preferably 2 to 15%, based on the weight of fats and oils to be decomposed. The amount of water is preferably 10 to 200%, particularly 20 to 100%, based on the weight of the fats and oils to be decomposed. The water may be any of distilled water, ion exchange water, tap water, well water and the like. If necessary, a buffer solution having a pH of 3 to 9 may be used so that the stability of the lipase can be maintained.
[0022]
The reaction temperature is preferably 20 to 70 ° C., particularly 30 to 50 ° C., which is a temperature at which the lipase is not deactivated and free fatty acids generated by decomposition do not become crystals. The reaction is desirably performed in the presence of an inert gas so that contact with air is avoided as much as possible.
[0023]
The hydrolysis reaction is managed by the decomposition rate represented by the following formula (1), and may be terminated when a predetermined decomposition rate is reached. As the degradation rate increases, the content of PUFA accumulated in undegraded glycerides increases.
[0024]
Formula (1): Decomposition rate (%) = acid value of reaction oil (AV) / saponification value of feedstock (SV) x 100
[0025]
The reaction oil obtained by the above decomposition reaction contains free fatty acids, undecomposed monoglycerides, diglycerides and triglycerides. By separating free fatty acids from these mixtures, it is possible to produce oils with high PUFA content.
[0026]
[Second step]
In the second step, the PUFA-rich oil obtained in the first step is hydrolyzed by acting two types of lipases, lipase acting on triglyceride and partial glyceride lipase. These two kinds of lipases need to act on oils and fats with a high PUFA content at the same time, so that a fatty acid with a high content of PUFA can be obtained in a high yield in a short time with a high decomposition rate.
[0027]
Examples of the lipase acting on the triglyceride used in the second step include the same lipases as those used in the first step.
[0028]
Partial glyceride lipase, which is another lipase used in the second step, is a lipase that hydrolyzes partial glycerides of monoglycerides and diglycerides but does not hydrolyze triglycerides. Partial glyceride lipase includes monoglyceride lipase or diglyceride lipase derived from animal organs such as rat small intestine and porcine adipose tissue; monoglyceride lipase derived from Bacillus sp. H-257 (J. Biochem., 127, 419-425, 2000), monoglyceride lipase derived from Pseudomonas sp. LP7315 (Journal of Bioscience and Bioengineering, 91 (1), 27-32, 2001), lipase derived from Penicillium cyclopium (J. Biochem, 87 (1), 205) -211, 1980), Penicillium camembertii U-150-derived lipase (J. Fermentation and Bioengineering, 72 (3), 162-167, 1991), etc., among which Penicillium (Penicillium) genus Lipase is preferred. Examples of commercially available products include “Lipase G“ Amano ”50” (Amano Enzyme).
[0029]
By using immobilized lipase as these two kinds of lipases, the degradation rate of oils with high PUFA content can be further increased, and the amount of lipase used can be reduced.
[0030]
The immobilization carrier used for immobilizing the lipase is preferably the same as the immobilization lipase used in the first step, and an immobilization carrier pretreated with a fat-soluble fatty acid or a derivative thereof should be used. Is also the same as the immobilized lipase in the first step. In addition, the immobilization conditions for the lipase acting on the triglyceride are the same as the immobilization lipase in the first step.
[0031]
The immobilization temperature of the partial glyceride lipase may be any temperature that does not cause enzyme inactivation, and is preferably 0 to 60 ° C, particularly preferably 5 to 45 ° C. The pH of the aqueous enzyme solution used for immobilization may be in a range that does not cause denaturation of the enzyme, and is preferably pH 3-9. In particular, when using a lipase whose optimum pH is acidic, the pH should be 4 to 6 in order to obtain the maximum activity. Moreover, as a buffer solution used for the enzyme aqueous solution, a general acetate buffer solution, phosphate buffer solution, Tris-HCl buffer solution, or the like can be used. The concentration of the partial glyceride lipase in the enzyme aqueous solution is desirably a sufficient concentration that is not more than the solubility of the enzyme from the viewpoint of immobilization efficiency. If necessary, the insoluble part may be removed by centrifugation, and the supernatant may be used. Moreover, the use ratio of the partial glyceride lipase and the immobilized carrier is preferably 0.05 to 10 parts by weight, particularly preferably 0.1 to 5 parts by weight, with respect to 1 part by weight of the immobilized carrier.
[0032]
In addition, it is preferable to reduce the enzyme water content by the same method as the immobilized lipase in the first step after adsorbing to the immobilized carrier for both of the two lipases.
[0033]
The amount of lipase used for the hydrolysis reaction in the second step can be appropriately determined in consideration of their activities, but both lipase acting on triglycerides and partial glyceride lipase are 1 with respect to the weight of fat with high PUFA content. -30%, especially 5-15% is preferred. Further, the amount of water is preferably 10 to 200%, particularly preferably 20 to 100%, based on the weight of the fat with high PUFA content. The water may be any of distilled water, ion exchange water, tap water, well water and the like. If necessary, a buffer solution having a pH of 3 to 9 may be used so that the stability of the lipase can be maintained.
[0034]
The reaction temperature is preferably 20 to 60 ° C., particularly 30 to 50 ° C., which is a temperature at which the lipase is not deactivated and free fatty acids generated by decomposition do not become crystals. The reaction is desirably performed in the presence of an inert gas so that contact with air is avoided as much as possible.
[0035]
The hydrolysis reaction in the second step is also controlled by the decomposition rate represented by the above formula (1), and may be terminated when the predetermined decomposition rate is reached. As the degradation rate increases, the production of fatty acids containing high concentrations of PUFA increases.
[0036]
The reaction oil obtained by the above decomposition reaction contains undegraded glycerides in addition to a fatty acid mixture containing high PUFA content fats and oils. By separating the fatty acid component from these mixtures, it is possible to obtain a PUFA-rich fatty acid. That is, after removing the aqueous phase containing lipase, glycerin, etc. from the reaction mixture, the free fatty acid fraction is fractionated by known fractionation means such as chromatography, molecular distillation, liquid-liquid distribution, crystal fractionation, deoxidation method and the like. As a result, a PUFA-rich fatty acid can be obtained.
[0037]
【Example】
[Immobilized enzyme production method]
10 g of Duolite A-568 (Rohm & Hass) was stirred in 100 mL of 0.1 mol / L sodium hydroxide aqueous solution for 1 hour. Then, it was washed with 100 mL of distilled water for 1 hour, and equilibrated with 100 mL of 500 mM phosphorus buffer (pH 7) for 2 hours. Thereafter, the pH was equilibrated with 100 mL of 50 mM phosphorus buffer (pH 7) twice for 2 hours. Thereafter, filtration was performed to recover the carrier, followed by ethanol replacement with 50 mL of ethanol for 30 minutes. After filtration, 50 mL of ethanol containing 10 g of ricinoleic acid was added and ricinoleic acid was adsorbed on the carrier for 30 minutes. Thereafter, the carrier was recovered by filtration, and then washed four times with 50 mL of 50 mM phosphorus buffer (pH 7), ethanol was removed, and the carrier was recovered by filtration. Thereafter, the mixture was contacted with 200 mL of a 10% solution of lipase (Lipase AY “Amano” 30G, Amano Enzyme) acting on a commercially available triglyceride for 4 hours for immobilization. After filtration, the immobilized enzyme was recovered and washed with 50 mL of 50 mM acetate buffer (pH 7) to remove unimmobilized enzyme and protein. All the above operations were performed at 20 ° C. The immobilization rate was found to be 95% from the difference between the residual activity of the enzyme solution after immobilization and the activity difference between the enzyme solution before immobilization. Thereafter, 40 g of rapeseed oil was added and stirred at 40 ° C. for 2 hours, followed by filtration to separate from rapeseed oil to obtain an immobilized enzyme. The immobilized enzyme thus obtained was washed with a PUFA-containing oil and fat which is a substrate that actually performs the reaction before use.
When immobilizing the partial glyceride lipase, the carrier was pretreated in the same manner using an acetate buffer (pH 5) instead of the phosphorus buffer (pH 7). After the treatment, the mixture was contacted with a 10% solution of a commercially available partial glyceride lipase (Lipase G “Amano” 50, Amano Enzyme) for 2 hours for immobilization. After washing, 40 g of soybean fatty acid was added, and dehydration under reduced pressure was performed at 40 ° C. Then, it filtered and isolate | separated from soybean fatty acid, and it was set as the fixed enzyme. The immobilized enzyme thus obtained was washed with a PUFA-containing oil and fat which is a substrate that actually performs the reaction before use.
[0038]
[First step]
5 g (dry weight) of the washed immobilized lipase AY (lipase acting on triglyceride) was weighed into a 100 mL four-necked flask. Thereto, 50 g of tuna deoxidized oil (DHA content 22%) and 30 g of distilled water were added, and the reaction was carried out at 40 ° C. for 78 hours with stirring under a nitrogen stream. The reaction solution was centrifuged (5,000 × g, 30 minutes), the acid value (AV) of the oil phase was measured, and the decomposition rate was calculated from equation (1). The decomposition rate was 73%. Thereafter, the oil phase part was subjected to molecular distillation to separate free fatty acids to obtain oils with high PUFA content. When fatty acid analysis was performed by GC, the DHA content was 46%, and the PUFA ratio was increased.
[0039]
Example 1
2 g each of lipase (lipase AY “Amano” 30G, Amano Enzyme) acting on triglyceride and partial glyceride lipase (Lipase G “Amano” 50, Amano Enzyme) were weighed in a 100 mL four-necked flask. Thereto were added 50 g of PUFA-rich oil (DHA content 46%) obtained in the first step and 30 g of distilled water, and the reaction was carried out at 40 ° C. for 71 hours while stirring under a nitrogen stream. Thereafter, the reaction solution is centrifuged (1,000 × g, 5 minutes), and the oil phase is developed on a TLC plate (developing solvent: chloroform / acetone / methanol = 93.5 / 4.5 / 2.0 vol%) and fractionated into each glyceride. After elution with ethyl acetate, the solvent was removed and the fatty acid composition was analyzed by GC. The results are shown in Table 1.
The ultimate decomposition rate was as high as 81.3%, and the DHA content in the fatty acid was also high as 45%.
[0040]
Example 2
Washed immobilized lipase AY (lipase acting on triglyceride) and washed immobilized lipase G (partial glyceride lipase) 5 g (dry weight) each were weighed into a 100 mL four-necked flask. Thereto were added 50 g of PUFA-rich oil (DHA content 46%) obtained in the first step and 30 g of distilled water, and the reaction was carried out at 40 ° C. for 68 hours with stirring under a nitrogen stream.
The ultimate degradation rate was as high as 90.1%, and the DHA content in fatty acids was also as high as 46%.
[0041]
Comparative Example 1
2 g of lipase (lipase AY) acting on triglyceride was weighed into a 100 mL four-necked flask. Thereto were added 50 g of PUFA-rich oil (DHA content 46%) obtained in the first step and 30 g of distilled water, and the reaction was carried out at 40 ° C. for 71 hours while stirring.
The ultimate decomposition rate was as low as 44.2%, and a high decomposition rate could not be achieved.
[0042]
Comparative Example 2
2 g of partial glyceride lipase (Lipase G) was weighed into a 100 mL four-necked flask. Thereto were added 50 g of PUFA-rich oil (DHA content 46%) obtained in the first step and 30 g of distilled water, and the reaction was carried out at 40 ° C. for 71 hours while stirring under a nitrogen stream.
The ultimate decomposition rate was as low as 24.3%, and a high decomposition rate could not be achieved.
[0043]
Comparative Example 3
2 g of lipase (lipase AY) acting on triglyceride was weighed into a 100 mL four-necked flask. Thereto were added 50 g of PUFA-rich oil (DHA content 46%) obtained in the first step and 30 g of distilled water, and the reaction was carried out at 40 ° C. for 47 hours with stirring under a nitrogen stream. Thereafter, 2 g of partial glyceride lipase (lipase G) was added, and the reaction was further carried out for 24 hours (a total of 71 hours).
The ultimate decomposition rate was as low as 73.9%, and a high decomposition rate could not be achieved.
[0044]
[Table 1]

[0045]
【The invention's effect】
According to the production method of the present invention, a fatty acid containing a high concentration of PUFA can be obtained very efficiently and in a high yield.

Claims (4)

The manufacturing method of the highly unsaturated fatty acid high content fatty acid containing the following 1st process and 2nd process.
1st process: After making lipase which acts on a triglyceride act on highly unsaturated fatty acid fats and oils and hydrolyzing, it isolate | separates a free fatty acid and obtains the fats and oils which raised the ratio of highly unsaturated fatty acids 2nd process: 1st A process for obtaining a fatty acid containing a highly unsaturated fatty acid at a high concentration by hydrolyzing a glyceride lipase that acts on triglycerides and a partial glyceride lipase on the highly unsaturated fatty acid-rich oil obtained in the process.   The production method according to claim 1, wherein the lipase used in the first step is an immobilized lipase. Two lipases used in the second step, the manufacturing method of Ah Ru請 Motomeko 1 or 2, wherein an immobilized lipase.   The production method according to claim 2 or 3, wherein the immobilized lipase is obtained by immobilizing lipase on an immobilization carrier pretreated with a fat-soluble fatty acid or a derivative thereof.